Enzyme-based electrochemical sensors are widely used in the detection of analytes in clinical, environmental, agricultural and biotechnological applications. Analytes that can be measured in clinical assays of fluids of the human body include, for example, glucose, lactate, cholesterol, bilirubin and amino acids. Levels of these analytes in biological fluids, such as blood, are important for the diagnosis and the monitoring of diseases.
Enzyme-based electrochemical sensors are devices in which a signal from an analyte-concentration-dependent biochemical reaction is converted into a measurable optical or electrical signal. Amperometric, enzyme-based biosensors typically employ two or three electrodes, including at least one measuring or working electrode and one reference electrode. The measuring or working electrode is composed of a non-corroding carbon or a metal conductor and is connected to the reference electrode via a circuit, such as a potentiostat. In three electrode systems, the third electrode is a counter-electrode. In two electrode systems, the reference electrode also serves as the counter-electrode.
The working electrode typically includes a sensing layer (also referred to herein as a “reagent layer”) in direct contact with the conductive material of the electrode. The sensing layer may include an enzyme, an enzyme stabilizer such as bovine serum albumin (BSA), and a cross-linker that crosslinks the sensing layer components. Alternatively, the sensing layer may include an enzyme, a polymeric mediator, and a cross-linker that crosslinks the sensing layer components, as in a “wired-enzyme” biosensor.
Upon passage of a current through the working electrode, a redox enzyme is electrooxidized or electroreduced. The enzyme is specific to the analyte to be detected, or to a product of the analyte. The turnover rate of the enzyme is typically related (preferably, but not necessarily, linearly) to the concentration of the analyte itself, or to its product, in the test solution.
The electrooxidation or electroreduction of the enzyme is often facilitated by the presence of a redox mediator in the solution or on the electrode. The redox mediator assists in the electrical communication between the working electrode and the enzyme. The redox mediator can be dissolved in the fluid to be analyzed, which is in electrolytic contact with the electrodes, or can be applied within a coating on the working electrode in electrolytic contact with the analyzed solution. The coating is preferably not soluble in water, though it may swell in water. Useful devices can be made, for example, by coating an electrode with a film that includes a redox mediator and an enzyme where the enzyme is catalytically specific to the desired analyte, or its product. In contrast to a coated redox mediator, a diffusional redox mediator, which can be soluble or insoluble in water, functions by shuttling electrons between, for example, the enzyme and the electrode. In any case, when the substrate of the enzyme is electrooxidized, the redox mediator transports electrons from the substrate-reduced enzyme to the electrode; when the substrate is electroreduced, the redox mediator transports electrons from the electrode to the substrate-oxidized enzyme.
Enzyme-based electrochemical sensors have employed a number of different redox mediators such as monomeric ferrocenes, quinoid-compounds including quinines (e.g., benzoquinones), nickel cyclamates, and ruthenium ammines. For the most part, these redox mediators have one or more of the following limitations: the solubility of the redox mediators in the test solutions is low, their chemical, light, thermal, or pH stability is poor, or they do not exchange electrons rapidly enough with the enzyme or the electrode or both. Additionally, the redox potentials of many of these reported redox mediators are so oxidizing that at the potential where the reduced mediator is electrooxidized on the electrode, solution components other than the analyte are also electrooxidized; in other cases they are so reducing that solution components, such as, for example, dissolved oxygen are also rapidly electroreduced. As a result, the sensor utilizing the mediator is not sufficiently specific. Sensors employing the redox polymers of the invention address these deficiencies.
Various biosensors have been designed to operate partially or wholly in a living body. Indeed, clinical use of such biosensors has been a significant step toward helping diabetic patients achieve tight control over their blood glucose levels.
In an example of an amperometric, enzyme-based glucose biosensor, the sensor utilizes glucose oxidase, which catalyzes the oxidation of glucose by oxygen in a sample of body fluid and generates gluconolactone and hydrogen peroxide, whereupon the hydrogen peroxide is electrooxidized and correlated to the concentration of glucose in the sample (Thom-Duret et al., Anal. Chem. 68, 3822 (1996); and U.S. Pat. No. 5,882,494 of Van Antwerp et al., filed on Aug. 28, 1995). In another example of an amperometric, enzyme-based, glucose biosensor, a polymeric redox mediator “wires” the reaction center of glucose oxidase to an electrode and catalyzes the electrooxidation of glucose to gluconolactone. The principle and the operational details of such a “wired-enzyme” biosensor have been described (Csoregi, et al., Anal. Chem. 1994, 66, 3131; Csoregi, et al., Anal. Chem. 1995, 67, 1240; Schmidtke, et al., Anal. Chem. 1996, 68, 2845; Schmidtke, et al., Anal. Chem. 1998, 70, 2149; and Schmidtke, et al., Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 294).
Enzyme-immobilized mediators allow electron transport between an enzyme active site and an electrode surface by shortening the electron tunneling steps. The term “wired enzyme” refers to enzymes with covalently attached redox mediators. The enzyme is in effect wired by the mediator to an electrode. The wired enzymes are able to transfer redox equivalents from the enzyme's active site through the mediator to an electrode.
The wired-enzyme principle resulted in subsequent development of enzyme-immobilizing redox polymers. These polymers effectively transfer electrons from glucose-reduced GOx flavin sites to polymer-bound redox centers. A series of chain redox reactions within and between polymers transfer the equivalents to an electrode surface. The redox enzyme and wire are immobilized by cross-linking to form three-dimensional redox epoxy hydrogels. A large fraction of enzymes bound in the three-dimensional redox epoxy gel are wired to the electrode.
In in vivo systems employed to monitor glucose, the electrochemical sensor may be inserted into a blood source, such as a vein or other blood vessel, for example, such that the sensor is in continuous contact with blood and can effectively monitor blood glucose levels. Further by way of example, the electrochemical sensor may be placed in substantially continuous contact with bodily fluid other than blood, such as dermal or subcutaneous fluid, for example, for effective monitoring of glucose levels in such bodily fluid. Relative to discrete or periodic monitoring, continuous monitoring is generally more desirable in that it may provide a more comprehensive assessment of glucose levels and more useful information, such as predictive trend information, for example. Subcutaneous continuous glucose monitoring is also desirable for a number of reasons, one being that continuous glucose monitoring in subcutaneous bodily fluid is typically less invasive than continuous glucose monitoring in blood.
The FREESTYLE NAVIGATOR continuous glucose sensor (Abbott Diabetes Care Inc., Alameda, Calif., USA) is a subcutaneous, electrochemical sensor, which operates for three days when implanted at a site in the body. This sensor is based on WIRED ENZYME sensing technology, as described above in which “wired” enzyme electrodes are made by connecting enzymes to electrodes through crosslinked electron-conducting redox hydrogels (“wires”). In FREESTYLE NAVIGATOR, glucose oxidase is “wired” with polyelectrolytes having electron relaying [Os(bpy)2Cl]+/2+ redox centers in their backbones. Hydrogels are formed upon cros slinking the enzyme and its wire on electrodes.
The operation and performance of an amperometric biosensor, such as those just described, may be complicated at high rates of analyte flux. For example, at high rates of glucose flux, an amperometric glucose biosensor may be kinetically overwhelmed, such that the relationship between the concentration of glucose in a sample fluid and the response from the biosensor becomes non-linear. This kinetic problem may be solved by the interposition of an analyte-flux-limiting membrane between the sample fluid and the sensing layer of the biosensor, as described in the above-mentioned U.S. patent Application Publication No. U.S. 2003/0042137 A1 of Mao et al. Still, the development of analyte-flux-limiting membranes, such as glucose-flux-limiting membranes, has not been without its challenges. Many known membranes have proved difficult to manufacture and/or have exhibited properties that limit their practical use, such as practical use in a living body.
The FREESTYLE NAVIGATOR sensor also comprises an analyte-restricting membrane, which may be a glucose-restricting membrane, disposed over the sensing layer. (See, e.g., U.S. Patent Application Publication No. 2003/0042137 A1 of Mao et al. filed May 14, 2002). The membrane is a bio-compatible polymer used to coat the outer surface of the sensing layer.
Further development of manufacturing techniques and methods, as well as analyte-monitoring devices, systems, or kits employing the same, is desirable.